CN111363377B - Nucleic acid dye and preparation method and application thereof - Google Patents

Nucleic acid dye and preparation method and application thereof Download PDF

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CN111363377B
CN111363377B CN202010177951.1A CN202010177951A CN111363377B CN 111363377 B CN111363377 B CN 111363377B CN 202010177951 A CN202010177951 A CN 202010177951A CN 111363377 B CN111363377 B CN 111363377B
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CN111363377A (en
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周杰
郑慧敏
马晓峰
谈雪良
钱近春
夏继波
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Suzhou Youyi landi Biotechnology Co.,Ltd.
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Abstract

The invention discloses a nucleic acid dye, a preparation method and application thereof, which are used for common gel imaging dyeing of nucleic acid electrophoresis, in particular to a novel nucleic acid dye which can be simultaneously suitable for agarose gel electrophoresis and polyacrylamide gel electrophoresis, especially can be used for blue light imaging, and relates to the preparation and application of the dye. The novel nucleic acid dye is developed, integrates good dyeing effect, high safety, very low nucleic acid migration influence rate and wide applicability, and can be used for blue light imaging. The defect that the existing nucleic acid dye cannot be applied to polyacrylamide gel electrophoresis due to poor dyeing effect is effectively solved, and the problems that the existing dye is easy to produce tailing and the like during running electrophoresis before being used are particularly solved.

Description

Nucleic acid dye and preparation method and application thereof
Technical Field
The invention is used for common gel imaging dyeing of nucleic acid electrophoresis, in particular to a novel nucleic acid dye which can be simultaneously suitable for agarose gel electrophoresis and polyacrylamide gel electrophoresis, especially can be used for blue light imaging, and relates to preparation and application of the dye.
Background
Nucleic acid electrophoresis is an important means for nucleic acid research, and is an essential component of techniques such as nucleic acid probe, nucleic acid amplification, and sequence analysis. Nucleic acid electrophoresis typically uses both agarose gels and polyacrylamide gels. Agarose is chain polysaccharide prepared by separating agar, and many agaroses are twisted with each other to form rope-shaped agarose bundles according to the action of hydrogen bonds and other forces to form large-mesh-type gel, which is suitable for separating and purifying nucleic acid fragments with the length of 200bp-50kB and can distinguish DNA fragments with the difference of 100bp; the polyacrylamide gel is a high polymer with very high density, has molecular sieve effect, concentration effect and charge effect, and has the best separation effect on small fragment DNA (5 bp-500 bp).
Common nucleic acid electrophoresis dyes are EB, SYBR Green, SYBR Gold, GelRed, GelGreen, and the like. The excitation wavelengths of different nucleic acid dyes are different, and at present, gel imagers commonly used in nucleic acid electrophoresis are divided into two types of bands, namely an ultraviolet gel imaging system and a blue light gel imager (or blue light gel cutting instrument). Ultraviolet long-time irradiation has certain harm to nucleic acid (DNA) and experimenters, so that future nucleic acid dyes tend to be safe and nontoxic blue-light nucleic acid dyes.
The existing nucleic acid dye, such as the existing gelgreen dye, is safe and non-toxic, has high sensitivity, but cannot be applied to polyacrylamide gel electrophoresis. The reasons for this are: polyacrylamide gel density is a more densely packed three-dimensional polymer. The highly dense polymer structure makes it difficult for fluorescent dye as nucleic acid gel dye to permeate into nucleic acid molecules in different size ranges and different concentration ranges, so that the nucleic acid dyeing effect is poor. In addition, the existing dyes have many limitations in application, for example, tailing is easy to generate during running electrophoresis of the prior dyes, namely, the influence on the mobility of DNA is large, the true level of DNA migration cannot be ensured, and thus, the experimental result has deviation. This effect on mobility, the same band shift will be significantly biased.
Disclosure of Invention
The invention discloses a nucleic acid dye and a preparation method and application thereof; compared with the existing products, the compound serving as the nucleic acid dye not only maintains the special high sensitivity, but also is safer and nontoxic, particularly solves the tailing phenomenon in the existing nucleic acid electrophoresis test, and can be particularly applied to agarose gel electrophoresis and polyacrylamide gel electrophoresis at the same time, and particularly can be imaged by blue light. The gel electrophoresis test result obtained by the compound of the invention under the same condition shows that the influence of the compound as a nucleic acid dye on the nucleic acid mobility is very little, and the accuracy and the sensitivity of the electrophoresis test can be ensured.
The invention adopts the following technical scheme:
the nucleic acid dye has the following chemical structural formula:
Figure 896420DEST_PATH_IMAGE001
Figure 344719DEST_PATH_IMAGE002
wherein a, b, c, y and z are independently selected from 0-15; r1Selected from hydrogen, alkyl, cycloalkyl or (CH)2)tSO3H and t are 1-5; r1Is selected from the group consisting of or (CH)2)xSO3 -X is 1-5; preferably, a and b are independently selected from 0-14, c, y and z are independently selected from 0-8; further preferably, a is selected from 1-12, b is selected from 0-12, c is selected from 0-5, y is selected from 1-3, and z is selected from 1-3;
t and x are independently selected from integers of 3-5;
R1is a substituent selected from hydrogen, alkyl or cycloalkyl; preferably, the number of carbon atoms in the alkyl or cycloalkyl group is less than 12;
b is a connecting bridge selected from the group consisting of-O [ (CH)2)n]O-、-(CH2)a1-[O-(CH2)y1]b1-[O-(CH2)z1]c1-、-(CH2)h-N-(CH2)k-、-[(CH2)n]O[(CH2)m]-one of the above; wherein n is 1 to 12, a11 to 12; y1 and z1 are independently selected from 1-5, preferably 2-3, b1 is 0-12, c1 is 0-5, h and k are independently selected from 1-12, and m is 1-12; or B is a polymethylene unit containing 1-24 carbons and optionally containing at least one heteroatom selected from N, O or containing a saturated 5-or 6-membered ring.
Y-Is an anion, including but not limited to halide or sulfonate ions (OTs)-）。
The ultraviolet gel imaging system is nucleic acid gel imaging equipment with higher popularity in the market at present, but ultraviolet long-time irradiation has certain harmfulness to nucleic acid and experimenters, so the future development trend is to use a safe blue light gel imaging system or a blue light gel cutting instrument. The dye is a nucleic acid dye for blue light imaging, is suitable for equipment such as a blue light gel imaging system or a blue light gel cutting instrument, and eliminates the harm of imaging equipment to nucleic acid and experimenters.
Nucleic acid dyes such as SYBR Green, GelGreen, etc. are available. SYBR Green can enter living cells due to small molecular weight and is combined into DNA of human cells to mutate nucleic acid, so that carcinogenic hidden danger exists for users, and the application market of the SYBR Green can be gradually reduced due to the potential harm; the GelGreen has large molecular weight, so that the dispersion and tailing phenomena are easy to occur in agarose electrophoresis, the polyacrylamide gel is used as a high polymer with higher density in a three-dimensional space, the dye is difficult to permeate into the polyacrylamide gel, so that the nucleic acid dyeing effect is poor, and the silver dyeing method for dyeing the polyacrylamide nucleic acid gel causes serious pollution to the environment and also seriously harms the human health due to the use of a large amount of silver nitrate and formaldehyde. The invention designs a new dye structure, eliminates the carcinogenic hidden trouble, also solves the phenomenon that the target strip in the original dye is easy to disperse, trailing and can not be separated, and simultaneously, the dye can easily permeate into high polymer with high density such as polyacrylamide gel.
The invention discloses application of the nucleic acid dye in nucleic acid blue light gel imaging; or the application of the nucleic acid dye as a nucleic acid blue light gel imaging dye; or the application of the nucleic acid dye in preparing blue light gel imaging reagents.
The invention discloses a preparation method of the nucleic acid dye, which comprises the following steps:
(1) taking 2- (methylthio) benzothiazole as a raw material, and carrying out substitution reaction to obtain an intermediate I;
(2) preparing an intermediate II by using 4-methylquinoline and bromo-polyethylene glycol-carboxylic acid as raw materials;
(3) taking the intermediate I and the intermediate II as raw materials to prepare an intermediate III;
(4) reacting the intermediate III with a diamine compound to prepare a nucleic acid dye;
the chemical structural formula of the intermediate I is as follows:
Figure 858877DEST_PATH_IMAGE003
、
Figure 785244DEST_PATH_IMAGE004
the chemical structural formula of bromo-polyethylene glycol-carboxylic acid is as follows:
Figure 130775DEST_PATH_IMAGE005
the chemical structural formula of the intermediate II is as follows:
Figure 749975DEST_PATH_IMAGE006
the chemical structural formula of the intermediate III is as follows:
Figure 548167DEST_PATH_IMAGE007
Figure 278225DEST_PATH_IMAGE008
the chemical structure of the diamine compound is as follows:
H2N-B-NH2。
the substituent in the preparation method is the same as that in the chemical structural formula of the nucleic acid dye.
The invention discloses a nucleic acid gel imaging method, which comprises the following steps of mixing the nucleic acid dye and an agarose solution, preparing agarose gel, putting the agarose gel into an electrophoresis tank for sample loading, carrying out electrophoresis, and finally imaging to finish nucleic acid gel imaging.
The invention discloses a nucleic acid gel imaging method, which comprises the following steps of loading a sample into polyacrylamide gel, then carrying out electrophoresis, immersing the gel subjected to electrophoresis into a staining solution, and finally imaging to finish nucleic acid gel imaging; the staining solution contains the nucleic acid dye.
The invention discloses a nucleic acid gel imaging method, wherein agarose gel or polyacrylamide gel is the prior art, the specific gel imaging process is also the prior art, the invention creativity lies in that the nucleic acid dye is disclosed, the nucleic acid dye is not only suitable for an agarose gel system, but also suitable for a polyacrylamide gel system, and particularly solves the problems that the existing dye is easy to generate the phenomenon that the dispersion tailing of a target strip cannot be separated and the nucleic acid mobility is influenced, and the nucleic acid gel imaging method is safe and nontoxic to human bodies and environments.
Compared with ultraviolet, blue light is the development trend of nucleic acid imaging, and the method is suitable for a blue light gel imaging system or a blue light gel cutting instrument. Compared with the common nucleic acid dye in the market, the invention can be used for agarose gel dyeing and polyacrylamide nucleic acid gel dyeing, solves the problems that part of the dye is easy to generate the phenomena that the target band is dispersed, trailing and cannot be separated, and the nucleic acid mobility is influenced, and is safe and nontoxic to human bodies and the environment.
Drawings
FIG. 1 is a mass spectrum of compound number 1 of Table 1 of the present invention;
FIG. 2 is a mass spectrum of compound number 2 of Table 1 of the present invention;
FIG. 3 is a mass spectrum of compound number 3 of Table 1 of the present invention;
FIG. 4 is a mass spectrum of compound number 5 of Table 1 of the present invention;
FIG. 5 is a mass spectrum of compound number 6 of Table 1 of the present invention;
FIG. 6 is an agarose gel of the compound of Table 1, number 1, of the present invention, a conventional GelGreen dye;
FIG. 7 is an agarose gel of inventive compound of Table 1, number 12;
FIG. 8 is a polyacrylamide gel diagram of a conventional UE Page GelRed dye;
FIG. 9 is a gel diagram of polyacrylamide of compound number 1 of Table 1 according to the present invention;
FIG. 10 is a polyacrylamide gel of inventive compound number 12 of Table 1;
FIG. 11 is a polyacrylamide gel of inventive compound number 5 of Table 1.
Detailed Description
EXAMPLE one preparation of nucleic acid dyes
1. A500 mL reaction flask was charged with 25 g of 2- (methylthio) benzothiazole, 30.8 g of methyl p-toluenesulfonate, mechanically stirred, heated to 60 ℃ and followed by TLC until the reaction was complete (developing solvent: acetonitrile: water = 5: 1); after the reaction is finished, adding 300 mL of ethyl acetate, heating to 75 ℃, and refluxing for 3 hours; then cooling to room temperature, filtering, washing the filter cake once with ethyl acetate, filtering, and drying the filter cake in vacuum to obtain 47 g of an intermediate I product.
Figure 478263DEST_PATH_IMAGE009
Wherein:
2- (methylthio) benzothiazole:
Figure 206047DEST_PATH_IMAGE010
methyl p-toluenesulfonate:
Figure 1789DEST_PATH_IMAGE011
intermediate I:
Figure 269959DEST_PATH_IMAGE012
2. in a 250 mL reaction flask was added 11.76 g 4-methylquinoline, 28 g bromo-tripeleneglycol-carboxylic acid, 10 mL o-dichlorobenzene, heated to 120 ℃ and followed by TLC until the reaction was complete (developing solvent: acetonitrile: water = 5: 2); after the reaction was complete, the reaction mixture was cooled to room temperature, ethyl acetate was added, washed twice, filtered and dried in vacuo to give 30 g of intermediate II.
Figure 590082DEST_PATH_IMAGE013
Wherein:
4-methylquinoline:
Figure 551085DEST_PATH_IMAGE014
bromo-tripethylene glycol-carboxylic acid:
Figure 527131DEST_PATH_IMAGE015
intermediate II:
Figure 333413DEST_PATH_IMAGE016
3. a500 mL reaction flask was charged with 27 g of intermediate I, 30 g of intermediate II, 50 mL of dimethylformamide DMF, 22mL of triethylamine TEA and TLC tracked to the end of the reaction (developing solvent: dichloromethane: methanol: glacial acetic acid = 5: 1: 0.1); after the reaction was completed, DMF was pumped off by vacuum pump, dissolved with acetonitrile, added dropwise to ethyl acetate to precipitate a solid, filtered, and the solid was vacuum dried for 48 hours to obtain 31 g of intermediate III.
Figure 773622DEST_PATH_IMAGE017
Wherein:
intermediate III:
Figure 639947DEST_PATH_IMAGE018
4. 200 mg of intermediate III, 3 mL of DMF and 150 uL of TEA were added to a 25 mL reaction flask, stirred for 15 minutes in an ice-water bath, 130mg of 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate TsTu (added in portions of 40mg +30mg +20mg +20mg at 5 minutes intervals) was added, and TLC was followed until the reaction of the intermediate III was complete (developing solvent: dichloromethane: methanol: glacial acetic acid = 5: 1: 0.1); 10uL of ethylenediamine is diluted to 100 uL with DMF and added into the reaction flask (added in portions according to 50 uL +30 uL +20 uL) and supplemented with one drop of triethylamine, and TLC is followed until the reaction is finished (developing agent: dichloromethane: methanol: glacial acetic acid = 5: 1: 0.1); after the reaction is finished, draining DMF, washing once with ethyl acetate, purifying with an alumina column, wherein the eluent is acetonitrile: water = 98: 2, collecting, spin-drying and freeze-drying to obtain a final product of 46 mg, which is shown as a compound of a number 1 in the table 1.
Figure 713262DEST_PATH_IMAGE020
In this example, the substitution reaction of step (1) is carried out in the presence of a sulfonic acid compound, such as methyl p-toluenesulfonate, to obtain an intermediate, a product which is an inner salt or a complex with a sulfonate ion as an anion. Further, the product taking the sulfonate ion as the anion is subjected to halogen ion replacement to obtain the product taking the halogen ion as the anion, namely the nucleic acid dye. The halide ion exchange may be carried out in a halide salt solution, a conventional technique.
Example two by replacing the starting material, other nucleic acid dyes in table 1 can be prepared, specifically by routine replacement.
For example, the ethylenediamine in the step (4) of the example is replaced by the compound of formula (i), or the compound of formula (ii), the compound of formula (iii), and the rest is unchanged, to obtain the compounds of numbers 2, 3, and 4 in table 1, wherein the chemical formulas of the compound of formula (i), the compound of formula (ii), and the compound of formula (iii) are as follows: h2N(CH2CH2O)3CH2CH2NH2、H2NCH2CH2OCH2CH2NH2、H2NOCH2CH2ONH2。
For example, the ethylenediamine in the example step (4) is replaced by the compound of formula 1 or the compound of formula 2, and the rest is not changed, to obtain the compound of number 7 and number 8 in table 1, wherein the compound of formula 1 and the compound of formula 2 have the following structural formulas:
Figure 243863DEST_PATH_IMAGE021
、
Figure 812247DEST_PATH_IMAGE022
for example, the bromo-tripentaethylene glycol-carboxylic acid in step (2) of the example was replaced with the compound of formula (iv), with the remainder unchanged, to give the compound of number 5 of table 1, wherein the compound of formula (iv) is as follows:
Figure 497306DEST_PATH_IMAGE023
the final product is subjected to anion replacement according to a conventional method to obtain dyes with different anion coordination, for example, the product of the first example is subjected to conventional replacement in a saturated sodium chloride aqueous solution or a sodium iodide aqueous solution, and is subjected to conventional stirring, concentration and filtration to obtain a solid which is a dye with chloride ion or iodide ion as an anion:
Figure 910970DEST_PATH_IMAGE024
Figure 60192DEST_PATH_IMAGE025
the other compounds in table 1 can be prepared according to conventional techniques on the basis of the above.
The methyl p-toluenesulfonate prepared in step (1) in the example was replaced with 20.2g of 1, 3-propanesultone, and the remainder was changed to give the compound No. 12.
TABLE 1 fluorescent nucleic acid dyes
Figure 737161DEST_PATH_IMAGE027
Figure 706254DEST_PATH_IMAGE029
Figure 923609DEST_PATH_IMAGE031
Figure 927337DEST_PATH_IMAGE033
Figure 775207DEST_PATH_IMAGE035
Figure 497175DEST_PATH_IMAGE037
Figure DEST_PATH_IMAGE039
Figure DEST_PATH_IMAGE041
Figure DEST_PATH_IMAGE043
Figure DEST_PATH_IMAGE045
Figure DEST_PATH_IMAGE047
Figure DEST_PATH_IMAGE049
The mass spectrograms of the compounds with the numbers 1, 2, 3, 5 and 6 in the table 1 are respectively shown in the attached figures 1 to 5.
The solid dye of the present invention is weighed, added to water and stirred to completely dissolve the dye to obtain a dye solution for the following gel imaging. In the nucleic acid gel imaging method disclosed by the invention, agarose gel or polyacrylamide gel is the prior art, the specific gel imaging process is also the prior art, and the creativity of the invention is to disclose the nucleic acid dye.
EXAMPLE three applications of the dyes according to the invention
(I) preparation of the dyes according to the invention
Weighing 5 mg of the nucleic acid dye of the invention, and adding diH2O100. mu.L was completely dissolved and OD was measured to 10000X, and DIH was used as it is2Diluting O to 1x to obtain working concentration, for example, adding 2.5 μ L into 25 mL gel to obtain 1x working concentration; the existing dyes are formulated as well.
Secondly, the specific experimental method for solving the dispersion tailing phenomenon in the agarose gel electrophoresis of the product of the invention is as follows:
1. weighing 0.6 g of agarose powder into a conical flask;
2. adding 60 mL of 1xTAE into the conical flask;
3. heating in a microwave oven, repeatedly boiling for three times to fully dissolve, wherein the volume of the final solution is 60 mL;
4. respectively measuring 25 mL of agarose solution in a measuring barrel, respectively pouring the agarose solution into two conical flasks, adding 2.5 mu L of a control group Biotium GelGreen (41005) dye solution into one conical flask, adding 2.5 mu L of the dye solution of the invention into one conical flask, shaking the conical flasks to uniformly mix the dye and the agarose solution, and respectively pouring the agarose solution into prepared gel devices;
5. gelling at room temperature for 1h (or standing at room temperature for 20 min until the gel does not shake, placing in a refrigerator at 4 deg.C, and standing for 15 min to solidify);
6. placing the coagulated agarose gel into an electrophoresis tank filled with electrophoresis solution (1 xTAE), and sequentially and conventionally loading 5 mu L of nucleic acid marker (conventional substance) in control group gel (GelGreen) and experimental group gel (the dye of the invention);
7. opening an electrophoresis tank switch after the sample loading is finished, setting the voltage to be 160V, setting the time to be 30 min, and starting electrophoresis;
8. after the electrophoresis is finished, taking out agarose, placing the agarose on a blue-light gel imaging system and a blue-light gel cutting instrument, photographing, storing pictures under a corresponding folder, and obtaining a result, namely fig. 6, in each picture, from left to right in a sample loading sequence: UE 100bp; UE 2000bp; 3, UE 5000 bp; UE 1 kb; 5, UE 15000 bp; YS 1 kb; takara 1 kb; 8. vazyme 1 kb.
As a result:
1. compared with a control group (left in FIG. 6, Gelgreen), the compound of the invention (right in FIG. 6, numbered 1 in the table) has lighter background color and clear strip;
2. compared with a control group, the large-section strips can be obviously separated, and the phenomenon of dispersion and tailing cannot occur;
3. the invention can effectively solve the phenomenon of dispersion and tailing in the existing product.
The same gel imaging procedure was used to replace compound No. 1 of table 1 with compound No. 12 of table 1 to obtain a gel chromatogram, see fig. 7, wherein the lanes are from left to right: 1.UE 2000bp 5 ul; 2, UE 15000bp 5 ul; DS 2000bp 10 ul; DS 15000bp 10 ul; takara 2000bp 10 ul; 6.takara 15000bp 5 ul.
(III) the specific experimental method of the product applied to the polyacrylamide gel electrophoresis is as follows:
1. adding water and standing for 20 min after the gel-making plate of the gel device is assembled, checking the sealing property of the device, and pouring clean water to prepare 2 pieces of 5% non-denatured PAGE gel when the sealing property is good;
2. in a 50 mL test tube, H was added sequentially2O9.4 mL, 30% Acrylamid 2.5mL, 5XTBE 3.0 mL finally 10% AP 0.11 mL and TEMED 0.010 mL were added and mixed (H)2O, 30% acrylamide and 5xTBE are prepared in advance, and 10% AP and TEMED are added before glue pouring is needed);
3. adding the mixed non-denatured glue to ensure that the glue making plate is completely filled with liquid, and inserting a comb (paying attention to the front and back sides, and the smooth side of the comb is close to the side of the glue making plate with the thick inside);
4. solidifying for 1h at room temperature to completely solidify the glue;
5. loading 5 mu L of sample;
6. the electrophoresis buffer solution is 1xTBE, the voltage is set to be 100V, the electrophoresis time is 60 min, and the electrophoresis is started;
7. preparing a dye soaking solution: taking 2 containers, respectively adding 100 mL of 0.1M NaCl solution (10 mL of 1M NaCl is mixed with 90 mL of deionized water), respectively adding 30 μ L of control dye UE Page gel Red (S2005) and the dye of the invention into the 100 mL of solution, and respectively and uniformly mixing;
8. carefully taking the gel off the glass plate after electrophoresis is finished, and putting the gel into a bubble dyeing machine for dyeing for 30 min;
9. after dyeing is finished, the blue-light gel imaging system, the blue-light gel cutting instrument and the ultraviolet gel imaging system are respectively placed, photographing is carried out, and pictures are stored under corresponding folders.
The control group UE Page GelRed dye-stained PAGE gel was visible only under UV and could not be imaged under blue light, see FIG. 8, lanes from left to right: 1.UE 100bp, 2.UE 2000pb, 3.Takara 100bp, 4.Takara 500bp, 5.Takara 2000bp, 6. RNA.
The dye-stained PAGE gel of the present invention (compound number 1 in Table 1) was imaged under blue light, as shown in FIG. 9, lanes from left to right: 1.UE 1kb 200ng, 2.UE 1kb 100ng, 3.UE 1kb 50ng, 4.UE 1kb 25ng, 5.UE 100bp, 6.UE 2000bp, 7.UE 5000bp, 8.UE 1kb, 9.UE 15000 bp.
As a result:
compared with a control group, the dye can dye PAGE gel, does not bring the problems of nucleic acid variation and the like caused by long-time irradiation under ultraviolet, does not cause any influence on a target band under blue light, and has clear bands.
The dye is replaced by the existing gelgreen dye, and the same polyacrylamide gel electrophoresis test is carried out, so that the strip cannot be observed no matter blue light or ultraviolet light, the imaging effect is very poor, and the strip cannot be observed.
Replacing compound No. 1 in Table 1 with compound No. 12 in Table 1, and performing the same gel imaging method to obtain PAGE gel chromatogram shown in FIG. 10, wherein the lane is from left to right: 1.UE 100bp 5 ul; 2, UE 2000bp 5 ul; DS 50bp 5 ul; takara 100bp 5 ul; 5.takara 2000bp 5 ul.
The same gel imaging method was used to replace the compound No. 1 in Table 1 with the compound No. 5 in Table 1 to obtain a PAGE gel chromatogram, see FIG. 11, lane: 1 from left to right, Takara 2000bp 1 ul; takara 2000bp 5 ul; takara 2000bp 10 ul; takara 2000bp 1 ul; takara 2000bp 5 ul; takara 2000bp 10 ul; UE 100bp 5 ul; UE 2000bp 5 ul; 9, UE 5000bp 5 ul; UE 1kb 5 ul; 11, UE 15000bp 5 ul; genstar 1kb 5 ul; takara 1kb 5 ul; vzayme 1kb 5 ul.
In conclusion, the novel nucleic acid dye is developed, integrates good dyeing effect, high safety, very low nucleic acid migration influence rate and wide applicability, and can be used for blue light imaging. The defect that the existing nucleic acid dye such as gelgreen dye cannot be applied to polyacrylamide gel electrophoresis due to poor dyeing effect is effectively overcome, and the problems that the existing dye is easy to produce tailing and the like during running electrophoresis during dyeing before use are particularly solved, namely the influence on the mobility of DNA is large, and the true level of DNA migration cannot be guaranteed, so that the experimental result has deviation, and the influence on the mobility and the same strip displacement can cause obvious deviation.

Claims (5)

1. The nucleic acid dye has the following chemical structural formula:
Figure 171795DEST_PATH_IMAGE001
Figure 761039DEST_PATH_IMAGE002
Figure 478460DEST_PATH_IMAGE003
Figure 542843DEST_PATH_IMAGE004
Figure 763740DEST_PATH_IMAGE005
Figure 992727DEST_PATH_IMAGE006
。
2. use of the nucleic acid dye of claim 1 in blue-light gel imaging of nucleic acids.
3. Use of the nucleic acid dye of claim 1 as a blue-light gel imaging dye for nucleic acids.
4. Use of the nucleic acid dye of claim 1 in the preparation of a blue light gel imaging agent.
5. The method for preparing the nucleic acid dye according to claim 1, comprising the steps of:
(1) taking 2- (methylthio) benzothiazole as a raw material, and carrying out substitution reaction to obtain an intermediate I;
(2) preparing an intermediate II by using 4-methylquinoline and bromo-polyethylene glycol-carboxylic acid as raw materials;
(3) taking the intermediate I and the intermediate II as raw materials to prepare an intermediate III;
(4) reacting the intermediate III with a diamine compound to prepare a nucleic acid dye;
the chemical structural formula of the intermediate I is as follows:
Figure 931864DEST_PATH_IMAGE007
、
Figure 537289DEST_PATH_IMAGE008
the chemical structural formula of the intermediate II is as follows:
Figure 878272DEST_PATH_IMAGE009
the chemical structural formula of the intermediate III is as follows:
Figure 275231DEST_PATH_IMAGE010
Figure 436085DEST_PATH_IMAGE011
the chemical structure of the diamine compound is as follows:
H2N-B-NH2
wherein, the substituent R1An anion Y-Chemical structure B and the number of repeating units a, B, c,y, z are identical to the product nucleic acid dye of claim 1.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101142326A (en) * 2005-03-17 2008-03-12 百奥提姆股份有限公司 Methods of using dyes in association with nucleic acid staining or detection and associated technology
CN106008495A (en) * 2016-06-13 2016-10-12 苏州宇恒生物科技有限公司 Preparation of novel nucleic acid dye for polyacrylamide gel electrophoresis

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5401847A (en) * 1990-03-14 1995-03-28 Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5410030A (en) * 1993-04-05 1995-04-25 Molecular Probes, Inc. Dimers of unsymmetrical cyanine dyes containing pyridinium moieties
US8742114B2 (en) * 2011-11-26 2014-06-03 Laiqiang Ying Nucleic acid detections and methods of their use
CN110426442B (en) * 2016-06-13 2022-02-11 苏州优逸兰迪生物科技有限公司 Method for testing nucleic acid gel electrophoresis
CN110845862B (en) * 2017-12-29 2021-03-19 苏州优逸兰迪生物科技有限公司 Preparation method of fluorescent dye
CN108929559B (en) * 2018-07-03 2020-05-15 珠海黑马医学仪器有限公司 Novel blue light electrophoresis nucleic acid dye and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101142326A (en) * 2005-03-17 2008-03-12 百奥提姆股份有限公司 Methods of using dyes in association with nucleic acid staining or detection and associated technology
CN102942566A (en) * 2005-03-17 2013-02-27 百奥提姆股份有限公司 Dimeric and trimeric nucleic acid dyes, and associated systems and methods
CN106008495A (en) * 2016-06-13 2016-10-12 苏州宇恒生物科技有限公司 Preparation of novel nucleic acid dye for polyacrylamide gel electrophoresis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
《Supporting information for Thiazole orange-peptide conjugates:sensitivity of DNA binding to chemical structure》;Jay R. Carreon等;《organic letters》;20040123;第6卷(第4期);1-9 *
《Thiazole Orange−Peptide Conjugates:Sensitivity of DNA Binding to Chemical Structure》;Jay R. Carreon等;《organic letters》;20040123;第6卷(第4期);517-519 *

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